Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

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Significance

The brain is a remarkably complex organ and cholesterol homeostasis underpins brain function. It is known that cholesterol is not evenly distributed across different brain regions; however, the precise map of cholesterol metabolism in the brain remains unclear. If cholesterol metabolism is to be correlated with brain function it is essential to generate such a map. Here we describe an advanced mass spectrometry platform to reveal spatial cholesterol metabolism in situ at 400-µm spot diameter on 10-µm tissue slices from mouse brain. We mapped, not only cholesterol, but also other biologically active sterols arising from cholesterol turnover in both wild type and mice lacking cholesterol 24S-hydroxylase (CYP46A1), the major cholesterol metabolizing enzyme.

Abstract

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm ². It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.

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A normal brain function requires constant levels of cholesterol, and the need for constancy seems to be higher here than in any other organ. Nature has met this need by isolation of brain cholesterol by a highly efficient blood-brain barrier. As a low synthesis of cholesterol is present in the brain, a mechanism for compensatory elimination is required. A decade ago we made the unexpected finding that the favoured mechanism for this involves conversion into 24S-hydroxycholesterol, followed by diffusion over the blood-brain barrier. Recent studies by us and others on this new pathway have given new insights into the mechanisms by which cholesterol homeostasis is maintained in the brain. We recently demonstrated a flux of another oxygenated product of cholesterol, 27-hydroxycholesterol, in the opposite direction. The latter flux may be important for neurodegeneration, and may be the link between hypercholesterolaemia and Alzheimer's disease. An overview of the above studies is presented and the possibility that the cholesterol 24S-hydroxylase in the brain may be important for memory and learning and that it may be a new drug target is discussed.

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Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain.

Rahul Shah, Tiina Kotti, Anthony Russell … (2008)

Cholesterol 24-hydroxylase is a highly conserved cytochrome P450 that is responsible for the majority of cholesterol turnover in the vertebrate central nervous system. The enzyme is expressed in neurons, including hippocampal and cortical neurons that are important for learning and memory formation. Disruption of the cholesterol 24-hydroxylase gene in the mouse reduces both cholesterol turnover and synthesis in the brain but does not alter steady-state levels of cholesterol in the tissue. The decline in synthesis reduces the flow of metabolites through the cholesterol biosynthetic pathway, of which one, geranylgeraniol diphosphate, is required for learning in the whole animal and for synaptic plasticity in vitro. This review focuses on how the link between cholesterol metabolism and higher-order brain function was experimentally established.

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Author and article information

Journal

Journal ID (nlm-ta): Proc Natl Acad Sci U S A

Journal ID (iso-abbrev): Proc. Natl. Acad. Sci. U.S.A

Journal ID (hwp): pnas

Journal ID (pmc): pnas

Journal ID (publisher-id): PNAS

Title: Proceedings of the National Academy of Sciences of the United States of America

Publisher: National Academy of Sciences

ISSN (Print): 0027-8424

ISSN (Electronic): 1091-6490

Publication date (Print): 17 March 2020

Publication date (Electronic): 4 March 2020

Publication date PMC-release: 4 March 2020

Volume: 117

Issue: 11

Pages: 5749-5760

Affiliations

[1] ^aInstitute of Life Science, Swansea University Medical School , SA2 8PP Swansea, Wales, United Kingdom;

[2] ^bAdvion Limited, Harlow, Essex CM20 2NQ, United Kingdom;

[3] ^cDepartment of Ophthalmology and Visual Sciences, Case Western Reserve University , Cleveland, OH 44106;

[4] ^dCentre for Mass Spectrometry Imaging, Biomolecular Research Centre, Sheffield Hallam University , S1 1WB Sheffield, United Kingdom

Author notes

¹To whom correspondence may be addressed. Email: y.wang@ 123456swansea.ac.uk or w.j.griffiths@ 123456swansea.ac.uk .

Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 3, 2020 (received for review October 13, 2019)

Author contributions: Y.W. and W.J.G. designed research; E.Y. performed research; R.A., M.B., N.M., I.P., J.N., M.R.C., and O.W.H. contributed new reagents/analytic tools; E.Y., D.O.F.S., O.W.H., Y.W., and W.J.G. analyzed data; and E.Y., Y.W., and W.J.G. wrote the paper.